Silencing DC expression of CD40 in vivo by CD40 siIL. (A) Organ distribution of CD40 siIL. Mice were intravenously injected with 15 μg naked Cy3-siRNA, isotype IgG-coupled Cy3-siILs, NLDC-145-coupled Cy3-siILs, or with PBS. Liver, spleen, and kidney organs were collected at 20 minutes and 4 hours after siRNA injection. Frozen organ sections were analyzed using fluorescence microscopy. (B) In vivo CD40 gene silencing determined by flow cytometry. Mice were treated with siILs and control reagents as described in panel A. At 48 hours after treatment, splenic CD11c⁺ DCs and CD19⁺ B cells were isolated using magnetic-activated cell sorter beads. Cells were stained with Cy5PE-labed anti-CD40 mAb, and the expression of CD40 was detected by flow cytometry. (C) In vivo CD40 gene silencing was determined by quantitative PCR. Splenic CD11c⁺ DCs from panel B were used for extraction of total RNA. CD40 gene expression at the mRNA level was determined by quantitative PCR as described in “Quantitative polymerase chain reaction.” (D) In vivo CD40 gene silencing on day 12. Mice were treated with siILs as described in panel A. The splenic CD11c⁺ DCs were isolated on day 12 after siIL treatment. The CD40 expression was determined by flow cytometry as described in panel B. Data presented are representative of 4 independent experiments. Fluorescence-activated cell sorter data and PCR results are representative of 3 or 4 independent experiments with 3 or 4 mice enrolled in each group. *P < .05.